The present invention relates to a gene (polynucleotide) encoding a thermostable glucokinase, a recombinant vector comprising this gene, a transformant transformed with the recombinant vector, and a process for producing a thermostable glucokinase through the use of the transformant.
Glucokinase [EC.2.7.1.2] is an enzyme which plays an important role in vivo in the first step of the glycolytic pathway, i.e. introduction of ATP. It is also an industrially useful enzyme and it is employed in various test reagents that are used for measuring whether glucose is in a test composition and in the consumption of glucose in a sample.
Glucokinase shows a very high substrate specificity for glucose. Hexokinase [EC.2.7.1.1], which is known as an enzyme that catalyzes similar reactions, is a different enzyme that acts upon various saccharides as substrates. These two enzymes are also differ from each other in that they show low amino acid sequence homology.
The present inventors have found that glucokinase isolated from Bacillus stearothermophilus, which is a thermophilic microorganism, is highly stable and they have proposed a process for efficiently producing the same (Japanese Patent Application Laid-Open No. 127086/1981).
There have been reported other methods of expressing glucokinase genes in vivo such as in Japanese Patent Application Laid-Open No. 102195/1985, Japanese Patent Application Laid-Open No. 124390/1986, Japanese Patent Application Laid-Open No. 127880/1999, Published Japanese Translation of International Patent Publication No. 512762/1998, Japanese Patent Application Laid-Open No. 292485/1994, Published Japanese Translation of International Patent Publication No. 507084/1998, Published Japanese Translation of International Patent Publication No. 505498/1998, Published Japanese Translation of International Patent Publication No. 508398/1996, Domestic Re-publication of International Patent Publication No. 012343/1998, etc. However, the glucokinases disclosed in these publications are different from the enzyme described in the present invention in the points of stability, affinity for substrate, etc. In addition, the expression methods disclosed therein are not suitable for the large scale production through the isolation and purification and, therefore, are different from the process disclosed in the present invention.
The above-mentioned production process proposed by the present inventors makes it possible to obtain a glucokinase having a high heat stability and an excellent storage stability, and to easily purify the enzyme. However, much energy is needed in producing a thermostable glucokinase by the above-mentioned process, since the microorganism is cultured at a high temperature of generally 50 to 60xc2x0 C. In addition, there still remains an unsolved problem that this microorganism can produce glucokinase only in a small amount and thus it is difficult to produce glucokinase on a large scale.
The present invention aims at providing a gene manipulation material for genetic engineeringly producing thermostable glucokinase originating from a thermophilic microorganism Bacillus stearothermophilus in a large amount, and a process for producing thermostable glucokinase through the use of this material.
To meet these goals, the present inventors have conducted extensive studies and, as a result, succeeded in the isolation of a gene for thermostable glucokinase produced by the above-described Bacillus stearothermophilus and in the determination of the structure thereof. Furthermore, they have prepared a recombinant vector having the gene encoding thermostable glucokinase inserted into a vector DNA and shown that the thermostable glucokinase can be efficiently produced by culturing a bacterial strain capable of producing the thermostable glucokinase. The bacterial strain was constructed by introducing the recombinant vector into a strain belonging to, for example, the genus Escherichia, in a medium and the like, thereby completing the present invention.
Accordingly, the first embodiment of the present invention relates to a gene encoding thermostable glucokinase which comprises the amino acid sequence represented by SEQ ID NO:1, or a gene encoding thermostable glucokinase which comprises an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:1 by the deletion, substitution or addition of one or several amino acids.
The second embodiment of the present invention relates to a gene encoding thermostable glucokinase which has a DNA comprising the base sequence represented by SEQ ID NO:2, or a gene encoding thermostable glucokinase which comprises a base sequence derived from the base sequence represented by SEQ ID NO:2 by the deletion, substitution or addition of one or several bases.
Further, the third embodiment of the present invention relates to a recombinant vector comprising a gene according to the first or second embodiment of the present invention.
The fourth embodiment of the present invention relates to a transformant comprising the recombinant vector of the third embodiment of the present invention.
The fifth embodiment of the present invention relates to a process for producing thermostable glucokinase, which comprises culturing the transformant according to the fourth embodiment of the present invention in a medium and collecting the thermostable glucokinase from the culture.